Study of Rgmc regulation by iron levels, anemia, inflammation and hypoxia

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

AltitudeOmics: Red Blood Cell metabolic adaptation to high altitude hypoxia

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia  through  the so-called  oxygen-dependent  metabolic  regulation,  which  involves  the competitive  binding  of  deoxyhemoglobin  and  glycolytic  enzymes  to  the  N-terminal  cytosolic domain  of  band  3.  This  mechanism  promotes  the  accumulation  of  2,3-DPG,  stabilizing  the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the  Bohr  effect.  Despite  in  vitro  studies,  in  vivo adaptations  to  hypoxia  have  not  yet  been completely elucidated. Within  the  framework  of  the AltitudeOmics  study,  erythrocytes  were  collected  from  21 healthy volunteers at sea level, after exposure to high altitude (5260m) for 1, 7 and 16days, and following  reascent  after  7days  at 1525m.  UHPLC-MS  metabolomics  results  were  correlated  to physiological and athletic performance parameters. Immediate  metabolic  adaptations  were  noted as early as a few hours from ascending  to >5000m, and maintained for 16 days at high altitude.  Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide and sulphur/H2S metabolism. Metabolic adaptations were preserved one week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory.

Study of Rgmc regulation by iron levels, anemia, inflammation and hypoxia

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The role of acute ambient hypoxia in the regulation of myostatin

When exposed to chronic hypoxia by pathophysiological or environmental causes humans show muscle atrophy, challenging homeostasis and increasing mortality rate. Chronic hypoxia also presents with elevated myostatin peptide, a negative regulator of muscle size. This work induced acute hypoxia in healthy individuals; hypothesizing hypoxia would increase myostatin expression in both muscle and plasma in a concentration- and time-dependent manner. Hypoxia (1 % O2) reduced C2C12 myoblast migration and myotube size in vitro. Myotube atrophy was time-dependent, longer exposures showed greater atrophy. Intracellular myostatin peptide was decreased at every time point measured. Myostatin and downstream signalling pathways in muscle showed a high degree of percentage similarity between mouse and human, when amino acid sequences were directly compared. Healthy males (N = 8) were exposed to 20.9 % O2 or 11.9 % O2 for 2 hours. Following hypoxic exposure myostatin peptide was reduced in muscle but not plasma, relative to control conditions. A second cohort (N = 8) was exposed to 12.5 % O2 for 10 hours. Plasma myostatin was decreased following hypoxia, muscle myostatin trended towards increasing. A third cohort (N = 9; n = 8 lowlander, n = 1 Sherpa) was exposed to 10.7 % or 12.3 % O2 for 2 hours. Plasma myostatin was reduced at both concentrations with no difference between concentrations noted. In response to chronic hypoxia, individuals lose muscle mass. Counter to the hypothesis of an increase in myostatin in both muscle and plasma, here a consistent decrease in plasma myostatin following acute hypoxia is seen. Muscle myostatin shows a variable response, with decreasing intracellular expression seen following a 2 hour hypoxic exposure, and trends towards an increase following 10 hours of hypoxia. Decreases in plasma and muscle myostatin may represent myostatin’s movement towards peripheral compartments in these acute timeframes. Hypoxia alone is capable of altering myostatin in healthy individuals; the effects of hypoxia on myostatin appear to differ between the acute timeframes examined here and chronic exposures in environmental or disease models.

The hypoxia that developed in a microtidal estuary following an extreme storm produced dramatic changes in the benthos

Runoff from an extreme storm on 22 March 2010 led, during the next 3 months, to the formation of a pronounced halocline and underlying hypoxia in the upper reaches of the microtidal Swan–Canning Estuary. Benthic macroinvertebrates were sampled between January 2010 and October 2011 at five sites along 10 km of this region. By mid-April, the number of species, total density, Simpson’s evenness index and taxonomic distinctness had declined markedly, crustaceans had disappeared and the densities of annelids and molluscs had declined slightly. These faunal attributes (except Simpson’s index) and species composition did not recover until after the end of the hypoxia. The survival of annelids and loss of crustaceans in this period reflects different sensitivities of these taxa to severe environmental stress. The results emphasise that microtidal estuaries with long residence times are highly vulnerable to the effects of environmental perturbations, particularly during warmer periods of the year.

The hypoxia that developed in a microtidal estuary following an extreme storm produced dramatic changes in the benthos

Runoff from an extreme storm on 22 March 2010 led, during the next 3 months, to the formation of a pronounced halocline and underlying hypoxia in the upper reaches of the microtidal Swan–Canning Estuary. Benthic macroinvertebrates were sampled between January 2010 and October 2011 at five sites along 10 km of this region. By mid-April, the number of species, total density, Simpson’s evenness index and taxonomic distinctness had declined markedly, crustaceans had disappeared and the densities of annelids and molluscs had declined slightly. These faunal attributes (except Simpson’s index) and species composition did not recover until after the end of the hypoxia. The survival of annelids and loss of crustaceans in this period reflects different sensitivities of these taxa to severe environmental stress. The results emphasise that microtidal estuaries with long residence times are highly vulnerable to the effects of environmental perturbations, particularly during warmer periods of the year.

Hypoxia-induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype

Ischemia caused by coronary artery disease and myocardial infarction leads to aberrant ventricular remodeling and cardiac fibrosis. This occurs partly through accumulation of gene expression changes in resident fibroblasts, resulting in an overactive fibrotic phenotype. Long-term adaptation to a hypoxic insult is likely to require significant modification of chromatin structure in order to maintain the fibrotic phenotype. Epigenetic changes may play an important role in modulating hypoxia-induced fibrosis within the heart. Therefore, the aim of the study was to investigate the potential pro-fibrotic impact of hypoxia on cardiac fibroblasts and determine whether alterations in DNA methylation could play a role in this process. This study found that within human cardiac tissue, the degree of hypoxia was associated with increased expression of collagen 1 and alpha-smooth muscle actin (ASMA). In addition, human cardiac fibroblast cells exposed to prolonged 1% hypoxia resulted in a pro-fibrotic state. These hypoxia-induced pro-fibrotic changes were associated with global DNA hypermethylation and increased expression of the DNA methyltransferase (DNMT) enzymes DNMT1 and DNMT3B. Expression of these methylating enzymes was shown to be regulated by hypoxia-inducible factor (HIF)-1α. Using siRNA to block DNMT3B expression significantly reduced collagen 1 and ASMA expression. In addition, application of the DNMT inhibitor 5-aza-2'-deoxycytidine suppressed the pro-fibrotic effects of TGFβ. Epigenetic modifications and changes in the epigenetic machinery identified in cardiac fibroblasts during prolonged hypoxia may contribute to the pro-fibrotic nature of the ischemic milieu. Targeting up-regulated expression of DNMTs in ischemic heart disease may prove to be a valuable therapeutic approach.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.

Effect of remote ischaemic preconditioning in cardiac dysfunction and end-organ injury following cardiac surgery with cardiopulmonary bypass in children: A translational approach investigating clinical outcome and myocardial molecular biology

Congenital heart disease (CHD) is the most common birth defect, causing an important rate of morbidity and mortality. Treatment of CHD requires surgical correction in a significant percentage of cases which exposes patients to cardiac and end organ injury. Cardiac surgical procedures often require the utilisation of cardiopulmonary bypass (CPB), a system that replaces heart and lungs function by diverting circulation into an external circuit. The use of CPB can initiate potent inflammatory responses, in addition a proportion of procedures require a period of aortic cross clamp during which the heart is rendered ischaemic and is exposed to injury. High O2 concentrations are used during cardiac procedures and when circulation is re-established to the heart which had adjusted metabolically to ischaemia, further injury is caused in a process known as ischaemic reperfusion injury (IRI). Several strategies are in place in order to protect the heart during surgery, however injury is still caused, having detrimental effects in patients at short and long term. Remote ischaemic preconditioning (RIPC) is a technique proposed as a potential cardioprotective measure. It consists of exposing a remote tissue bed to brief episodes of ischaemia prior to surgery in order to activate protective pathways that would act during CPB, ischaemia and reperfusion. This study aimed to assess RIPC in paediatric patients requiring CHD surgical correction with a translational approach, integrating clinical outcome, marker analysis, cardiac function parameters and molecular mechanisms within the cardiac tissue. A prospective, single blinded, randomized, controlled trial was conducted applying a RIPC protocol to randomised patients through episodes of limb ischaemia on the day before surgery which was repeated right before the surgery started, after anaesthesia induction. Blood samples were obtained before surgery and at three post-operative time points from venous lines, additional pre and post-bypass blood samples were obtained from the right atrium. Myocardial tissue was resected during the ischaemic period of surgery. Echocardiographic images were obtained before the surgery started after anaesthetic induction and the day after surgery, images were stored for later off line analysis. PICU surveillance data was collected including ventilation parameters, inotrope use, standard laboratory analysis and six hourly blood gas analysis. Pre and post-operative quantitation of markers in blood specimens included cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), inflammatory mediators including interleukins IL-6, IL-8, IL-10, tumour necrosis factor (TNF-α), and the adhesion molecules ICAM-1 and VCAM-1; the renal marker Cystatin C and the cardiovascular markers asymmetric dymethylarginine (ADMA) and symmetric dymethylarginine (SDMA). Nitric oxide (NO) metabolites and cyclic guanosine monophosphate (cGMP) were measured before and after bypass. Myocardial tissue was processed at baseline and after incubation at hyperoxic concentration during four hours in order to mimic surgical conditions. Expression of genes involved in IRI and RIPC pathways was analysed including heat shock proteins (HSPs), toll like receptors (TLRs), transcription factors nuclear factor κ-B (NF- κ-B) and hypoxia inducible factor 1 (HIF-1). The participation of hydrogen sulfide enzymatic genes, apelin and its receptor were explored. There was no significant difference according to group allocation in any of the echocardiographic parameters. There was a tendency for higher cTnI values and inotropic score in control patients post-operatively, however this was not statistically significant. BNP presented no significant difference according to group allocation. Inflammatory parameters tended to be higher in the control group, however only TNF- α was significantly higher. There was no difference in levels of Cystatin C, NO metabolites, cGMP, ADMA or SDMA. RIPC patients required shorter PICU stay, all other clinical and laboratory analysis presented no difference related to the intervention. Gene expression analysis revealed interesting patterns before and after incubation. HSP-60 presented a lower expression at baseline in tissue corresponding to RIPC patients, no other differences were found. This study provided with valuable descriptive information on previously known and newly explored parameters in the study population. Demographic characteristics and the presence of cyanosis before surgery influenced patterns of activity in several parameters, numerous indicators were linked to the degree of injury suffered by the myocardium. RIPC did not reduce markers of cardiac injury or improved echocardiographic parameters and it did not have an effect on end organ function; some effects were seen in inflammatory responses and gene expression analysis. Nevertheless, an important clinical outcome indicator, PICU length of stay was reduced suggesting benefit from the intervention. Larger studies with more statistical power could determine if the tendency of lower injury and inflammatory markers linked to RIPC is real. The present results mostly support findings of larger multicentre trials which have reported no cardiac benefit from RIPC in paediatric cardiac surgery.

Impact of Hypoxia on the Community Structure of Benthic Macroinvertebrates of Lagos Lagoon, Nigeria

Study of the Lagos lagoon was conducted for two years to investigate the impact of hypoxia on the benthic macroinvertebrates. Water and benthic samples were collected monthly along the study stretch and analysed in a standard laboratory. Temporal variation in water physico-chemistry was largely controlled by rainfall pattern while the spatial variation was influenced by proximity to the Harbour as well as the pollution sources and types. A total of 3,159 individuals comprising three phyla, five classes, nineteen families and twenty three species were recorded. Iddo I, Iddo II, Ogudu and Agboyi study stations recorded very low individuals, but relatively high number of polychaetes. Benthic macro- invertebrate community was dominated by the molluscs. Margalef’s index of species richness ranged from 0.79 to 2.57 while Shannon-Wiener index ranged from 0.40 to 2.19. Species evenness index ranged from 0.29 to 0.80. There was generally low biodiversity indicating the stressed nature of the study area.

Physiological response of American bullfrog tadpoles to stressor conditions of capture and hypoxia

A rã-touro americana (Rana catesbeiana) recentemente denominada Lithobates catesbeianus é cria da com propósito comercial em várias regiões do Brasil. Situações estressantes tais como problemas de manejo, criação inadequada e alterações ambientais com consequente redução da imunidade são comuns em produções intensivas. A avaliação destas situações de estresse permite-nos detectar estes probemas e diminuir as injurias causadas pelo confinamento. O principal objetivo deste estudo foi utilizar os marcadores biológicos de cortisol, glicemia e dados hematológicos para avaliar a resposta de girinos de rã-touro submetidos aos mecanismos estressores de captura e hipóxia. Os animais foram distribuídosem três tratamentos: estresse por captura individual com puçá; estresse por captura em massa com puçá e estresse por captura por escoamento. Os resultados obtidos demostraram não haver diferenças estatisticamente significativas entre os parametros avaliados quando comparou-se os grupos com e sem exposição ao ar (normoxia e hipoxia). Com base nestes resultados pode-se concluir que os estímulos estressores avaliados não foram adequados para alterar os valores plasmáticos dos marcadores biológicos testados. Para o cortisol, isto ocorreu provavelmente em virtude da ação sinérgica deste hormônio e a tiroxina, que induz a metamorfose nestes animais.

Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors

O objetivo deste estudo foi avaliar a resposta fisiológica (cortisol, glicemia e parâmetros sanguineos) de girinos de rã-touro (Lithobates catesbeianus) em diferentes densidades e após exposição aérea. Os animais utilizados no experimento estavam entre os estágios 31 a 39, na fase de pró-metamorfose sendo testados 1 girino/L (Tratamento 1), 5 girinos/L (Tratamento 2) e 10 girinos/L (Tratamento 3), conduzidos em 3 réplicas simultâneas durante 12 dias. O sangue foi retirado por rompimento do vaso caudal na condição de Normóxia - N (tempo zero) e Hipóxia - H (tempo de 15 minutos de exposição ao ar). Foi observado um aumento nos valores de cortisol, aos 4 e 8 dias de exposição aérea retornando aos valores basais ao final do experimento, apesar de não haver diferenças significativas. A glicemia não apresentou diferenças significativas quanto aos estressores aplicados. Os parâmetros hematológicos da série branca, principalmente, o número de linfócitos, neutrófilos e eosinófilos mostraram diferença significativa aos 12 dias de experimentação quando comparados com o momento zero; concluindo-se que, em condições controladas, o adensamento de até 10 girinos/litro e a exposição aérea por 15 minutos não apresentou danos aos girinos de rã-touro durante o período experimental. O padrão de resposta a estes estímulos talvez seja expresso em outro nível hormonal (corticosterona).